【作者】 樊怡；

【导师】 马健飞；

【作者基本信息】 中国医科大学 ， 内科学， 2002， 硕士

【摘要】 前言 腹膜透析（PD）作为终末期肾衰竭（ESRF）替代治疗的有效手段之一，其长期有效的进行有赖于腹膜保持良好的透析效能。PD时腹膜持续暴露于酸性、高渗透压、高葡萄糖浓度的非生理性透析液，导致第一道生理屏障——腹膜间皮细胞（PMCs）发生显著的形态学改变：如细胞表面微绒毛消失、细胞增大甚至细胞剥脱，间皮细胞下基质增多，一些病人可出现基底膜增厚及间质中微血管壁增厚等糖尿病样病变，认为是暴露于极高葡萄糖浓度（83～234mmol／L）的透析液所致。上述结构改变会影响腹膜的透析效能：腹膜的通透性增加，葡萄糖（Glc）吸收加快，导致超滤（UF）下降。可见高糖是造成PMCs损害进而导致透析效能下降、超滤能力丧失的主要原因。因此有必要研究和探讨高糖对PMCs的损伤机制及PMCs对Glc的吸收机制，而PMCs的体外培养则为该研究提供了细胞模型。 Glc的摄取是己糖激酶（HKs）和细胞膜上葡萄糖转运蛋白（GLUTs）在功能上相互偶联的结果。HKs是在ATP和Mg²⁺存在的条件下，将经GLUTs易化扩散进入细胞的Glc磷酸化为6-磷酸葡萄糖，是Glc进入代谢途径的第一步反应。该反应一方面保证了细胞内外Glc的浓度梯度，使其连续不断的进入细胞内；同时也为Glc进入其它代谢途径提供了共同底物（6-磷酸葡萄糖）。因此细胞内HKs活性变化，决定了细胞对Glc的摄取。 已有实验证实：肾小球系膜细胞的HKs活性在糖尿病相关因素：蛋白激酶C（PKC）激活剂——佛波酯（PMA、PDD）的作用下上调，并伴有细胞对Glc净利用的增加，从而参与了糖尿病肾病的发生发展。而PMCS在PD时暴露于高糖透析液中，是否这种暴露也刺激了 HKS在PMCS的表达呢？因此有必要研究作为 GIC代谢关键酶之一的HKS是否参与了上述病理过程。 关于HKS在PMCS的表达国内外尚未见报道。本实验利用原代培养的PMCS，主要研究糖尿病相关因素：高糖JMA及PDD等PKC激活剂对PMCS的HKS活性的调节，检测HKS活性与0 摄取的相关性；并观察经典型PKCkPKC )抑制剂一e976对HKS活性的影响，为进一步探讨如何减少 GIC的吸收从而增加 UF提供新的理论依据。 实验材料 l、实验动物：雄性SD大鼠 2、实验用细胞：原代培养的腹膜间皮细胞 3、细胞培养、传代及鉴定的相关试剂 4、检测己糖激酶活性及葡萄糖含量的相关试剂 实验方法 1、胰蛋白酶消化法进行大鼠PMCS的原代培养 2、间接免疫荧光法用于PMCS的鉴定 3在一磷酸葡萄糖脱氢酶偶联比色法检测总HK活性 4、观察高糖、佛波酯及CPKC抑制剂对HKS活性的调节 5上色法检测细胞培养液中葡萄糖减少的量，观察细胞对葡萄糖的净利用能力。 ·2· 实验结果 ＼胰蛋白酶消化20 dn、先弃消化液再用胎牛血清厂BS)吹打收集细胞及培养液含FBS浓度为10％的条件下，原代培养所获细胞的贴壁率高、纯度及生长良好，5－sd可达融合，形成铺路石样外观。经免疫荧光鉴定：细胞角蛋白抗体在胞浆内呈绿色荧光，证实培养的细胞是腹膜间皮细胞。 2、PMCs的基础 HKs活性为（4．80。0．39）U／g蛋白。 3、葡萄糖对HKs活性的诱导呈浓度依赖性 葡萄糖浓度为 2．5％和 4．25％的培养液刺激 PMCS 24 h后，＊征活性分别提高65．9 ％及so啪(P＜O．05人而渗透压对照组 4．25％的甘露醇对 HKS的诱导为 3．2％(P＞0．05人 4JMA对HKS活性的诱导呈剂量和时间依赖性，同时伴有GIC净利用的增加。 ①PMCs暴露于PM人浓度为0．01 pmo＊L、0．lpmol／L及二卜moUL的培养液24 h后，HKs活性分别提高30．7％J9．9％及99．二％（P SO．00）。 ②PMCs暴露于…moVL PMA后，分别在 lh、3 h、6 h、12 h。18 h、24 h、30 h及 36 h检测 HKS活性。明显的诱导出现在刺激后12 h门8．5％，P＜0．0（）厂 HKS活性的最大诱导时间出现在刺激后24 h（pp．4％，P＜O．00）。 ③PMCs暴露于 1卜moVL PMA 18 h后，细胞对0c的净利用明显增加小．6nunol，P＜o．00＊，24卜培养液内的＊c基本消耗一半K，P北．皿1X与未经＊＊A刺激的细胞对比mc净利用差异显著（P＜o．05）。 5、佛波酯对HKS活性的诱导具有PKC特异性 PMCs暴露于 PDD浓度为0．001 po＊L、0．of pmo＊L及0．l ·3·1llllol／L的培养液24 h后，HKS活性分别提高25．3％52．5％及83．6畅(P＜O．01人而相同条件下4a－P＊D对＊Ks活性的诱导分别为6．6％3．4助及4．9呢（P＞O．05）。 6、Glib976抑制了 PMA对 HKS活性的诱导 PMCs暴露于浓度为10 pmo＊L二0 pmo＊L、30 pmo＊L、40umo＊L及50 umol／L的 e976预处理30 dn后，再予三卜mol／LPMA培养24 h，H征活性分别抑制了 14．6％、26．8％、34．2畅、44．2％及54．9％(P＄0．001人 讨 论 一、腹膜间皮细胞的体外培养 PMCS是构成?更多还原

【Abstract】 IntroductionPeritoneal Dialysis ( PD) is an adecpuate and effective renal replacement therapy for end - stage renal failure. The longevity of PD depends on the long - term preservation of the peritoneum as a dialytic membrane. During dialysis, the Peritoneal Mesothelial Cells (PMCs) acting as a biological barrier is continuously exposed to unphysiologic peritoneal dialysate because of its low pH, hyperosmolality and high glucose content. The unphysiologic dialysate resulted in significant morphological changes; this include increase of the PMCs volume, loss of microvilli, denudation of PMCs, increased accumulation of extracellular matrix (ECM) under the PMCs. Thickening and diabeti-form reduplication of basement membrane of mesothelium and stroma blood vessels of peritoneum were encounted in some patients. The extremely high glucose concentration (83 to 234 mmol/L) in the dialysate was supposed to contribute to these changes. The changes in the structure of peritoneum may influence its dialysis efficiency, peritoneum function changed markedly: increased permeability, increased glucose absorption and decreased ultrafiltration ( UF) . Therefore highconcentration glucose (Glc) may severely damage the structure and functions of PMCs, which is considered to be the primary cause of the decline in UF and dialysis efficacy. It is necessary to study the mechanism of peritoneal glucose absorption and to investigate the mechanism by which high glucose damage PMCs during PD. The primary culture of PMCs will provide the cell model for the research.Glucose uptake and utilization are the functional coupling and combining effect of both glucose transporters ( GLUTs) and Hexoki-nases ( HKs). HKs take the central play in this process via phospho-rylating Glc to yield Glc -6 - phorsphate. By this mechanism, HKs keep the favorable downhill concentration gradient that permits facilitated Glc entry cells. In addition, they initiate all subsequent pathways of Glc utilization. So the change of HKs activity may determine the Glc utilization.The accomplished animal experiments showed that protein kinase C ( PKC ) induced the activity of HKs in primary cultured rat mesang-ial cells and increased net Glc utilization. This plays an important role in the progression of diabetic nephropathy. During PD, the PMCs are exposed to dialysates containing high concentration of glucose. Does this kind of exposure stimulate the expression of HKs in PMCs?The expression and regulation of HKs in PMCs have not been explored. The present study investigated the effect of high glucose and phorbal esters on HKs activity in primary cultured PMCs and the association between HKs and glucose uptake, which might provide the theories basis for how to reduce the absorption of glucose and increase UF.Materials1. Experimental animals: Albino male rats of the Sprague Dawley (SD) strain were used.2. Experimental cells; Rat peritoneal mesothelial cells were isolated by primary culture.3. Reagents relating to cell culture and identification.4. Reagents relating to measurement of hexokinases activity and glucose concentration.Methods1. Enzymatic disaggregation used for primary culture2. Immunofluorescent staining used for identification3. Standard G6PDH - coupled assay used for measurement of HKs4. Observing the regulating effect of each treatment factor5. Colorimetric assay used for measurement of net Glc utilizationResults1. Confluent mesothelial cells were obtained by limited treatment with trypsin, it took 5 to 8 days to reach confluency. Immunofluorescent staining of confluent cells demonstrated that all cells reacted positively to cytokeratin.2. The basic activity of HKs in rat PMCs of primary culture was (4. 80 +0.39) U/g protein.3. Glucose induced the expression of HKs in a dose - dependent manner. At concentration of 2. 5 % and 4. 25 % Glc, the increase in HKs activity were 65.9 % and 80 % ( P <0. 05) , respectively above the level in the control, the activity of HKs induced by 4.25 %更多还原